High intensity sweeteners possess a sweetness level that is many times greater than the sweetness level of sucrose. They are essentially non-caloric and are used widely in the manufacturing of diet and reduced calorie food. Although natural caloric sweetener compositions, such as sucrose, fructose, and glucose, provide a desirable taste to consumers, they have a caloric content. High intensity sweeteners, being essentially non-caloric, do not affect the blood glucose level and provide little or no nutritive value.
However, the high intensity sweeteners generally used as sugar (sucrose) substitutes possess taste characteristics different from those of sugar. The taste characteristics that differ from those of sugar may include the temporal profile of the sweet taste, maximal response, flavor profile, mouthfeel, and adaptation behavior. For example, the sweet taste of some high-potency sweeteners are slower in onset and longer in duration than the sweet taste produced by sugar and thus change the taste balance of a food composition. Because of these differences, the use of high-potency sweeteners to replace a bulk sweetener such as sugar, in a food or beverage, may cause an imbalance in the temporal and/or flavor profile. If the taste profile of high-potency sweeteners could be modified to impart desired taste characteristics, high-potency sweeteners could be used to provide more desirable taste characteristics to low calorie beverages and food products.
On the other hand, high-potency sweeteners may have some cost and functional advantages compared to sugar. There is significant competition among sugar and non-sugar sweeteners in the soft drink industry, in countries where the use and production of high-potency sweeteners is permitted, and in countries with overvalued sugar prices.
At present high intensity sweeteners are used worldwide. They can be both synthetic and natural origin.
Examples of synthetic sweeteners include, but are not limited to, sucralose, potassium acesulfame, aspartame, alitame, saccharin, neohesperidin dihydrochalcone, cyclamate, neotame, dulcin, suosan, N—[N-[3-(3-hydroxy-4-methoxyphenyl)propyl]-L-α-aspartyl]-phenylalanine 1-methyl ester, N—[N-[3-(3-hydroxy-4-methoxyphenyl)-3-methylbutyl]-L-α-aspartyl]-L-phenylalanine 1-methyl ester, N—[N-[3-(3-methoxy-4-hydroxyphenyl)propyl]-L-α-aspartyl]-L-phenylalanine 1-methyl ester, salts thereof, and the like.
Examples of natural high intensity sweeteners include, but are not limited to, Stevioside, Rebaudioside A, Rebaudioside B, Rebaudioside C, Rebaudioside E, Rebaudioside F, Steviolbioside, Dulcoside A, Rubusoside, mogrosides, brazzein, neohesperidin dihydrochalcone, glycyrrhizic acid and its salts, thaumatin, perillartine, pernandulcin, mukuroziosides, baiyunoside, phlomisoside-I, dimethyl-hexahydrofluorene-dicarboxylic acid, abrusosides, periandrin, carnosiflosides, cyclocarioside, pterocaryosides, polypodoside A, brazilin, hernandulcin, phillodulcin, glycyphyllin, phlorizin, trilobtain, dihydroflavonol, dihydroquercetin-3-acetate, neoastilibin, trans-cinnamaldehyde, monatin and its salts, selligueain A, hematoxylin, monellin, osladin, pterocaryoside A, pterocaryoside B, mabinlin, pentadin, miraculin, curculin, neoculin, chlorogenic acid, cynarin, Luo Han Guo sweetener, and siamenoside.
High intensity sweeteners can be derived from the modification of natural high intensity sweeteners by, for example, fermentation, contact with enzymes, or derivatization.
At present about twelve high intensity sweeteners are used worldwide. These are acesulfame-K, alitame, aspartame, cyclamate, glycyrrhizin, neohesperidin dihydrochalcone (NHDC), saccharin, Stevioside, sucralose, thaumatin, neotame, and Rebaudioside A.
The high intensity sweeteners can be grouped into a few different generations. The first generation, represented by cyclamate, glycyrrhizin and saccharin, has a long history of use in food. The second generation includes acesulfame-K, aspartame, NHDC and thaumatin. Alitame, neotame, sucralose, Stevioside, and Rebaudioside A belong to the third generation.
The standard sweetening power associated with each high intensity sweetener is given in TABLE 1. However, when they are used in blends, the sweetening power can change significantly.
TABLE 1SweetenerSweetness powerSaccharose (sucrose)1Acesulfame-K200Alitame2000Aspartame200Cyclamate30Glycyrrhizin50NHDC1000Saccharin300Stevioside200Rebaudioside A450Thaumatin3000Sucralose600
“Natural” and “organic” foods and beverages have become the “hottest area” in the food industry. The combination of consumers' desire, advances in food technology, and new studies linking diet to disease and disease prevention have created an unprecedented opportunity to address public health through diet and lifestyle.
A growing number of consumers perceive the ability to control their health by enhancing their current health and/or hedging against future diseases. This creates a demand for food products with enhanced characteristics and associated health benefits, and creates a food and consumer market trend towards a “whole health solutions” lifestyle. The term “natural” is highly emotive in the world of sweeteners and has been identified as a term that is highly trusted by consumers, along with “whole grain”, “heart-healthy” and “low-sodium”. Among many consumers, the term “natural” is closely related to “healthier”. In this respect, natural high intensity sweeteners can have better commercial potential than artificial sweeteners.
Stevia rebaudiana Bertoni is a perennial shrub of the Asteraceae (Compositae) family native to certain regions of South America. The leaves of the plant contain diterpene glycosides in an amount ranging from about 10 to 20%. These diterpene glycosides are about 150 to 450 times sweeter than sugar. The leaves of the Stevia rebaudiana Bertoni plant have been traditionally used for hundreds of years in Paraguay and Brazil to sweeten local teas and medicines. The plant is commercially cultivated in Japan, Singapore, Taiwan, Malaysia, South Korea, China, Israel, India, Brazil, Australia, and Paraguay.
At present more than 230 Stevia species have been discovered, including one Stevia species that has significant sweetening properties. The plant has been successfully grown under a wide range of conditions from its native subtropics to the cold northern latitudes.
The extract of the Stevia rebaudiana plant contains a mixture of different sweet diterpene glycosides which have a single base, steviol, and differ by the presence of carbohydrate residues at positions C13 and C19. These glycosides accumulate in Stevia leaves and compose approximately 10%-20% of the total dry weight. Typically, on a dry weight basis, the four major glycosides found in the leaves of Stevia are Dulcoside A (0.3%), Rebaudioside C (0.6-1.0%), Rebaudioside A (3.8%) and Stevioside (9.1%). Other glycosides identified in Stevia extract include Rebaudioside B, C, D, E, and F, Steviolbioside and Rubusoside. Among steviol glycosides only Stevioside and Rebaudioside A are available on a commercial scale.
Steviol glycosides have zero calories and can be used wherever sugar is used. They are ideal for diabetic and low calorie diets. In addition, the sweet steviol glycosides possess functional and sensory properties superior to those of many high potency sweeteners.
The chemical structures of the diterpene glycosides of Stevia rebaudiana Bertoni are presented in FIGS. 2a-2k. 
The physical and sensory properties of steviol glycosides are well studied only for Stevioside and Rebaudioside A. Stevioside is about 210 times sweeter than sucrose, and Rebaudioside A is between 200 and 400 times sweeter than sucrose. Rebaudioside A is considered to have the most favorable sensory attributes of the four major steviol glycosides.
Amongst the sweet diterpenoid glycosides of Stevia, Rebaudioside D has been identified as the least bitter, and with the least persistent aftertaste. Impurities can significantly increase the bitterness of diterpenoid glycosides.
The glycosides can be extracted from leaves using either water or organic solvent extraction. Supercritical fluid extraction and steam distillation methods have also been described. Methods for the recovery of diterpene sweet glycosides from Stevia rebaudiana using supercritical CO2, membrane technology, and water or organic solvents, such as methanol and ethanol, may also be used.
Generally the production of an extract includes extraction of plant material with water or a water-organic solvent mixture, precipitation of high molecular weight substances, deionization, and decolorization, purification on specific macroporous polymeric adsorbents, concentration and drying.
All of the existing methods for the recovery of steviol glycosides involve the isolation and purification of a steviol glycoside from the initial plant extract, and do not include a method for the further treatment of residual solutions or the purification of minor compounds. Thus, there is a need for an efficient and economical method for the comprehensive retreatment of extract produced from the Stevia rebaudiana Bertoni plant.
Due to regulatory requirements, only materials containing more than 95% total steviol glycosides are allowed to be used as sweeteners for human consumption. The majority of commercially available Stevia rebaudiana raw extracts contain 80-90% total steviol glycosides. Hence further purification is necessary to obtain highly purified products with more than 95% total steviol glycoside content. Therefore, there is a need for a commercially viable process for enhancing steviol glycoside content in low purity steviol glycoside preparations, to the levels which allow their usage in food.
Among sweet glycosides existing in Stevia rebaudiana, only Stevioside and Rebaudioside A are available at moderate cost at <80% purity and at high cost at >80% purity. There are no commercial quantities of Rebaudiosides B, D, E, F and C available in the market.
Rebaudioside D (CAS No: 63279-13-0) is one of the sweet glycosides found in Stevia rebaudiana. Its isolation and purification is a very challenging task due to its low content in Stevia leaves. The average Rebaudioside D content in dry leaves ranges from about 0.01-0.20%. Moreover, many analytical techniques often fail to detect Rebaudioside D in Stevia leaves or steviol glycoside preparations, due to its low content.
Only recently have highly purified Rebaudioside D reference materials become available on the market. Due to the high cost of the purification techniques employed, the price of such materials makes it impractical to use them in any area other than analytical chemistry.
However, studies show that highly purified forms of Rebaudioside D possess a very desirable taste profile, almost lacking in bitterness and in the lingering licorice aftertaste typical for other steviol glycosides. These properties multiply the significance of Rebaudioside D and attract great interest for methods of preparation of highly purified forms of Rebaudioside D.
The methods of Rebaudioside D preparation described in the literature employ costly chromatographic techniques which are only applicable for laboratory or pilot scale production. There is no published data on the commercial isolation and purification of Rebaudioside D.
Sakamoto et al. describe a process of isolation of rebaudioside D from the glycosidic fraction of stevia leave methanolic extract prepared according to Kohda et al. Sakamoto I., Yamasaki Tanaka O. (1977), “Application of 13C NMR Spectroscopy to Chemistry of Natural Glycosides: Rebaudioside-C, a New Sweet Diterpene Glycoside of Stevia rebaudiana.” Chem. Pharm. Bull., 25(4), p. 844; Kohda H., Kasai R., Yamasaki K., Murakami K., Tanaka O. (1976), “New sweet diterpene glucosides from Stevia rebaudiana.” Phytochemisty, 15, p. 981. The process comprises recrystallization of a glycosidic fraction from methanol and further chromatography on silica gel. The described process employs solvent extraction and chromatographic techniques which are useful in laboratory and pilot scale, but have limited scale-up potential due to the high cost of the process and toxicity of the extraction solvents.
Hence, there is a need for a simple, efficient, and economical method for the production of high purity Rebaudioside D.
There is also a need for a commercially viable process for enhancing steviol glycoside content in low purity steviol glycoside preparations to the levels which allow their usage in food.
Any technological scheme which can effect the purification of a mixture of low purity steviol glycosides, into a mixture of highly purified steviol glycosides and highly purified Rebaudioside D, will have a certain advantage over the techniques currently known in the art.